epsilonproteobacterium Sulfurimonas gotlandica GD1Toceanrep.geomar.de/26985/1/mbo3153.pdf · doi:10.1002/mbo3.153 Abstract Epsilonproteobacteria have been found globally distributed

ORIGINAL RESEARCH

Impact of dissolved inorganic carbon concentrations andpH on growth of the chemolithoautotrophicepsilonproteobacterium Sulfurimonas gotlandica GD1T

Kerstin Mammitzsch, G€unter Jost & Klaus J€urgens

Sektion Biologische Meereskunde, Leibniz-Institut f€ur Ostseeforschung Warnem€unde, Seestraße 15, D-18119, Rostock, Germany

Keywords

Chemolithoautotrophy, DIC saturation,

epsilonproteobacterium, ocean acidification,

pelagic redox zones, pH, sulfurimonas.

Correspondence

Klaus J€urgens, Sektion Biologische

Meereskunde, Leibniz-Institut f€ur

Ostseeforschung Warnem€unde, Seestraße 15,

D-18119, Rostock, Germany. Tel: +49 (381)

5197 250; Fax: +49 (381) 5197 211;

E-mail: [email protected]

Funding Information

This research was funded by the German

Federal Ministry of Education and Research

(BMBF), joint research project BIOACID,

subproject 1.1.1.

Received: 8 August 2013; Revised: 8 October

2013; Accepted: 22 October 2013

MicrobiologyOpen 2014; 3(1): 80–88

doi: 10.1002/mbo3.153

Abstract

Epsilonproteobacteria have been found globally distributed in marine anoxic/

sulfidic areas mediating relevant transformations within the sulfur and nitrogen

cycles. In the Baltic Sea redox zones, chemoautotrophic epsilonproteobacteria

mainly belong to the Sulfurimonas gotlandica GD17 cluster for which recently a

representative strain, S. gotlandica GD1T, could be established as a model

organism. In this study, the potential effects of changes in dissolved inorganic

carbon (DIC) and pH on S. gotlandica GD1T were examined. Bacterial cell

abundance within a broad range of DIC concentrations and pH values were

monitored and substrate utilization was determined. The results showed that

the DIC saturation concentration for achieving maximal cell numbers was

already reached at 800 lmol L�1, which is well below in situ DIC levels. The

pH optimum was between 6.6 and 8.0. Within a pH range of 6.6–7.1 there was

no significant difference in substrate utilization; however, at lower pH values

maximum cell abundance decreased sharply and cell-specific substrate con-

sumption increased.

Introduction

The hypoxic areas of the oceans, that is where the oxygen

concentration is less than 2 mL L�1, are currently increas-

ing on a global scale (Stramma et al. 2008). The most

severe form of anoxia, with fatal consequences for higher

life, constitutes the development of hydrogen sulfide-con-

taining bottom waters. These so called “dead zones” have

expanded due to eutrophication in coastal ecosystem (D�ıaz

and Rosenberg 2008) but exist also in semi-enclosed basins

with reduced water circulation such as the Black Sea, Cari-

aco Basin, and the Baltic Sea. The Baltic Sea is one of the

largest hypoxic marine systems and it is intensely influ-

enced by anthropogenic activities (Conley et al. 2011).

At the interface between hydrogen sulfide and oxygen

and/or nitrate, different groups of sulfur oxidizing bacteria

play an important role in the detoxification of hydrogen

sulfide (Lavik et al. 2009). Bacterial chemolithoautotrophic

denitrification occurs when there is an interface between

sulfide and nitrate and has been shown to be an important

process for removal of both nitrogen and hydrogen sulfide

(Jensen et al. 2009; Lavik et al. 2009; Grote et al. 2012;

Bruckner et al. 2013). Chemoautotrophic denitrification is

a widely distributed metabolic route, inter alia, carried out

by members of the b-, c- and e-proteobacteria, as well asAquificales and some other bacterial and archeal groups,

which occur across a wide range of habitats (Mat�ej�u et al.

1992; Reysenbach et al. 2009; Shao et al. 2010). However,

80 ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. This is an open access article under the terms of

the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,

provided the original work is properly cited.

there is evidence that e-proteobacteria seem to dominate

this process in marine pelagic redox zones (Brettar et al.

2006; Lin et al. 2006; Grote et al. 2007), but are also wide-

spread in hydrothermal vents (Campbell et al. 2006). In

fact, these species are responsible for the majority of che-

moautotrophy in the redox zones, as they carry out 70–100% of the CO2 fixation in the pelagic redox gradients of

the Baltic and Black Seas (Grote et al. 2008; Glaubitz et al.

2009). Different groups of e-proteobacteria have been

found globally distributed in marine anoxic/sulfidic areas

(Grote et al. 2012; Rodriguez-Mora et al. 2013) and proba-

bly are also widespread in coastal anoxic zones with a

sulfide–nitrate interface. In the Baltic Sea redox zones,

e-proteobacteria mainly belong to the Sulfurimonas gotlan-

dica GD17 cluster (Grote et al. 2007). Grote et al. (2012)

recently studied a representative of this Sulfurimonas clus-

ter, named S. gotlandica GD1T (Labrenz et al. 2013), and

used genomic and physiological investigations to demon-

strate high-metabolic versatility and adaptations to pelagic

redox zones of this strain. The bacterium is known to

reduce nitrate to dinitrogen and to oxidize thiosulfate to

sulfate.

Thus, this group of e-proteobacteria fulfills an impor-

tant ecological role in the oxic–anoxic interface of the

Baltic Sea, being primarily responsible for hydrogen sul-

fide detoxification and nitrate removal.

Using S. gotlandica GD1T as a model organism for this

group (Labrenz et al. 2013), we were investigating abiotic

and biotic factors which regulate the growth and distribu-

tion of these bacteria in the environment. Although in

previous studies the utilization of different electron

donors and acceptors (Grote et al. 2012; Labrenz et al.

2013) and the impact on the distribution of this group in

the redox zone (Bruckner et al. 2013) was studied, we

examined here the effects of dissolved inorganic carbon

(DIC) concentration and pH on growth of S. gotlandica

GD1T.

The important role of chemolithoautotrophic e-proteo-bacteria in the sulfur and nitrogen cycle led us to ask

whether and how these bacteria are able to cope with

changes in DIC concentrations and pH which are pre-

dicted due to ocean acidification in marine environments.

According to the report of the Intergovernmental Panel

on Climate Change (IPCC), a decrease of 0.3 pH units is

predicted by the year 2100 and a decrease of 0.77 pH

units by the year 2300 (Caldeira and Wickett 2003). How-

ever, in the deeper anoxic zones of the central Baltic

basins the DIC concentration is already around

2 mmol L�1 and the pH is 7.1 (Beldowski et al. 2010;

Schneider 2011) and acidification impacts are therefore

assumed to be relatively small in the environment. Only

few studies have examined the impact of DIC and pH on

growth of chemolithoautotrophic bacteria, mostly with

focus on carbon concentrating mechanisms, and using

isolates derived from hydrothermal vent habitats (e.g.,

Dobrinski et al. 2005; Scott and Cavanaugh 2007). There-

fore, in this study we first investigated whether different

DIC concentrations and pH values have an influence on

growth of S. gotlandica GD1T. Second, in order to deduce

single regulating factors, we examined the influence of

different pH values not only for growth but also for sub-

strate utilization.

Material and Methods

Cultivation

Sulfurimonas gotlandica GD1T was grown in anoxic artifi-

cial brackish water with the following components:

95 mmol L�1 NaCl, 11.23 mmol L�1 MgCl, 2.28

mmol L�1 CaCl2, 2.03 mmol L�1 KCl, 10 mmol L�1

HEPES, 192 lmol L�1 KBr, 91 lmol L�1 H3BO3,

34 lmol L�1 SrCl2, 91 lmol L�1 NH4Cl, 9 lmol L�1

KH2PO4, and 16 lmol L�1 NaF. Resazurin served as the

redox indicator. To remove oxygen from the medium,

the deionized water used in medium preparation was

boiled for at least 10 min and then purged with N2 for at

least 45 min. After autoclaving the medium, vitamins

(Balch et al. 1979), trace elements SL10 (Widdel et al.

1983), selenite, and tungstate (Widdel and Bak 1992)

were added as supplements. Nitrate (1 mmol L�1) was

added as electron acceptor and thiosulfate (1 mmol L�1)

as electron donor. Both were prepared anaerobically and

afterward autoclaved. Although hydrogen sulfide is an

important substrate in situ and was shown to be utilized

by this strain (Grote et al. 2012), thiosulfate provides high

cell numbers as well and is better suitable for controlled

experimental investigations (Grote et al. 2012; Bruckner

et al. 2013). The substrate concentrations were added in

saturation for S. gotlandica GD1T, allowing exponential

growth for several days. As carbon source sodium bicar-

bonate (filter-sterilized), was provided at a concentration

of 2 mmol L�1. Because there is an equilibrium of

hydrogen carbonate, carbonate, and carbon dioxide (DIC

speciation), the carbon source will be named as DIC con-

centration. The distribution of the DIC speciation at pH

6.5 is 70.96% hydrogen carbonate, 28.96% carbon diox-

ide, and 0.07% carbonate, whereas at pH 8.0 the distribu-

tion of the DIC speciation is 95.59% hydrogen carbonate,

1.23% carbon dioxide, and 3.17% carbonate.

The bacterium was grown in batch culture at 15°C in

the dark and at a pressure of 2.5 bar (N2-atmosphere) in

all experiments. To determine the DIC saturation as well

as optimum pH range 250 mL bottles were used includ-

ing 50 mL headspace. In the experiments to examine sub-

strate utilization during chemolithoautotrophic growth,

ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 81

K. Mammitzsch et al. Impact of DIC and pH on S. gotlandica

600-mL bottles were used with 100-mL headspace. Bacte-

rial cell numbers were quantified by counting DAPI (4′,6-diamidino-2-phenylindol)-stained cells by epifluorescence

microscopy. The maximal cell numbers, which were

reached at the end of the exponential growth phase, rep-

resent both the yield of the culture (with respect to the

substrate concentrations) and the carrying capacity under

the given conditions, reflecting the efficiency of using the

available substrates and converting them into bacterial

biomass.

Chemical analysis

The pH was measured with a WTW microprocessor pH

meter pH 3000 and a WTW SenTix 61 pH electrode and

calibrated with standard buffer solutions (pH 4.01 and

6.87). All pH measurements are reported on the National

Bureau of Standards (NBS) scale. The pH was measured

at the beginning (marked as pHs) and end (marked as

pHe) of the incubation time. After preparing the medium

(including autoclaving and cooling) and adding the sub-

strates and the desired DIC concentration, a 20-mL sub-

sample was taken from the anoxic medium and its pH

adjusted to the desired value by addition of 0.1 mol L�1

hydrochloric acid at room temperature. The correspond-

ing amount of 1mol L�1 HCl was then calculated and

added to the medium, which was then inoculated with

the bacteria. Changes in pH caused by different tempera-

tures between the measurement and incubation tempera-

ture were calculated with the carbonate equilibration

model, CO2SYS (Lewis and Wallace 1998). Nitrate was

quantified colorimetrically at a wavelength of 540 nm

according to the spongy cadmium method, as described

by Jones (1984). Sulfate was determined turbidimetrically

by Ba precipitation in a procedure modified from that of

Tabatabai (1974). Here, to avoid the formation and pre-

cipitation of thiosulfate-derived zero-valent sulfur, the

samples were not acidified by citric acid. Thiosulfate was

analyzed with a modified method according to Zopfi

et al. (2004). The samples were derivatized with 3-(bro-

momethyl)-2,5,6-trimethyl-1H,7H-pyrazolo[1,2-a]pyraz-

ole-1,7-dione (also known as (mono)bromobimane) and

then measured by HPLC (Merck), consisting of a LiChro-

sphere 60RP select B column (125 9 4 mm, 5 lm). The

eluents were 0.25% acetic acid (v/v) and HPLC-grade

methanol. The methanol gradient was established as fol-

lows: 0 min: 0%, 1 min: 8%, 4.5 min: 10%, 7 min: 32%,

11 min: 32%, 18 min: 50%, 22 min: 100%, 24 min:

100%, 25 min: 0%, and 30 min: 0%. Thiosulfate was

detected by a fluorescence detector (excitation: 380 nm,

emission: 480 nm). Standards and reagent blanks were

prepared in N2-purged deionized water and analyzed as

described for the samples.

Experimental design

Estimation of DIC saturation for growth

Sulfurimonas gotlandica GD1T was grown in batch culture

at DIC concentrations ranging from 20 lmol L�1 to

2000 lmol L�1 and at a pHs between 7.0 and 7.5. DIC

concentration was not measured directly but instead

sodium bicarbonate was dissolved and then added to the

medium to obtain the desired final concentration. From

the theoretical equilibrium between CO2 compounds in

the medium and in the headspace it was calculated that a

maximum of 1.8% of the DIC was converted into CO2 gas

in the headspace. Previous experiments had shown that

during growth in batch culture and under the conditions

applied, S. gotlandica GD1T reaches stationary phase after

10–14 days (Grote et al. 2012; Bruckner et al. 2013). Thus,

final cell concentrations at this time represent the carrying

capacity for this strain under the given conditions. There-

fore, we took samples after 14 days and quantified cell

numbers by DAPI staining. Cell number at the beginning

of the incubation time was 2.0 9 105 cells mL�1.

The parameters for bacterial growth in relation to the

DIC concentration were estimated by a nonlinear regres-

sion according to the function B = Bmax 9 ([DIC]�S)/

(Kd + [DIC]�S) using dynamic fitting procedure by Sig-

maPlot 10.0 (Systat Software, Inc., San Jose, CA), where

B = cell abundance (cells mL�1), Bmax = maximum cell

abundance (cells mL�1), [DIC] = dissolved inorganic car-

bon concentration (lmol L�1), S = threshold (lmol L�1)

and Kd = half-saturation concentration (lmol L�1).

Effects of different pH values onchemolithoautotrophic growth

To identify the pH range allowing the chemolithoauto-

trophic growth of S. gotlandica GD1T, the bacteria were

cultivated within a pHs range of 6–9. Accordingly, a pHs

range �0.05 of the target pHs was established. HEPES

(10 mmol L�1) was used as the buffer based on its opti-

mum buffering capacity between pH 6.8 and 8.0. The pH

of the bacterial preculture medium was between 7.0 and

7.5. Bacteria were grown in batch culture for 14 days, with

the final cell number determined as described above. At the

end of the incubation, the pHe was controlled using the

same methods described above. Cell number at the begin-

ning of the incubation time was 2.5 9 105 cells mL�1.

Substrate utilization duringchemolithoautotrophic growth

After the pH range had been determined, which is suitable

for chemolithoautotrophic growth of S. gotlandica GD1T,

82 ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Impact of DIC and pH on S. gotlandica K. Mammitzsch et al.

substrate utilization was investigated at selected pH values

within this range. S. gotlandica GD1T was grown under the

same conditions as above, and the pH was measured at

the beginning (pHs) and end (pHe) of the experiments.

The experiments were conducted at pHs of 7.1 (the pH of

the Baltic Sea redox zones), and at pHs 6.6 (the critical

point at which the influence of pH on maximal cell num-

bers became visible before). Cell number at the beginning

of the incubation time was 2.7 9 105 cells mL�1.

Cell numbers, nitrate and thiosulfate consumption, and

sulfate production were quantified daily for 14 days,

although substrate utilization per bacterium was calcu-

lated only during late exponential growth (day 6–9).Nitrate was analyzed in 1 mL samples diluted 1:100, and

sulfate in undiluted 1 mL samples. Thiosulfate was mea-

sured in 25-lL samples centrifuged and diluted 1:10 prior

to derivatization with 50 lL of Monobromobimane-HE-

PES-EDTA-buffer. The derivative was diluted again 1:10

to obtain a thiosulfate concentration below 20 lmol L�1,

that is within the concentration range yielding the best

linear relationship. Sulfate was measured immediately,

whereas nitrate and thiosulfate samples were stored at

�20°C until analysis. All vials used for the analyses were

flushed with N2 to remove oxygen and to maintain the

samples as oxygen-free as possible. Negative controls

without bacteria had been previously performed and

revealed that purely chemical reactions can be ruled out

for changes in substrate concentrations at different pH

values(Bruckner et al. 2013).

Statistical tests were performed using an analysis of var-

iance (ANOVA) and an error probability of 5% followed

by a post hoc test (Tukey).

Results


Sulfurimonas gotlandica GD1T grew at the whole DIC

concentrations but final cell numbers increased with

increasing DIC concentration up to a saturation level at

around 800 lmol L�1 DIC. At this concentration maxi-

mal cell number of 2.8 9 107 � 3.4 9 106 cells mL�1

was reached (Fig. 1). According to the calculated dynamic

fitting procedure by SigmaPlot, half-saturation concentra-

tion in DIC for achieving maximal cell numbers was

132.6 lmol L�1, with a threshold concentration of

87.5 lmol L�1 DIC.


The optimum pHs range for S. gotlandica GD1T, as judged

from the final cell concentration, was between 6.7 and 8.0,

with no significant differences in the maximal cell numbers

of 1.7 9 107 � 2.9 9 106 cells mL�1 (ANOVA, P > 0.05)

(Fig. 2). At pHs values above 8.0 and below 6.5, cell num-

bers did not differ from the initial levels, indicating that no

growth occurred. At pHs 6.5, bacterial cell numbers

increased only slightly, resulting in a maximal cell number

of 3.3 9 106 � 1.5 9 106 cellsmL�1. The pH measure-

ments at the end of the experiment showed that the pH in

the range of 6.5 and 8.4 remained constant (�0.02) during

the experimental time whereas above and below these

points the pHe decreased by about 0.18–0.25.

Figure 1. Maximal cell numbers of Sulfurimonas gotlandica GD1T

under different DIC conditions. The three replicates at each DIC

concentration are shown separately. The bacterium was grown for

14 days in batch culture. Data are shown as a rectangular curve

(r2 = 0.96), corresponding to a half-saturation concentration of

132.6 lmol L�1 and a threshold concentration of 87.5 lmol L�1 DIC.

Figure 2. Influence of pH on maximal cell numbers of Sulfurimonas

gotlandica GD1T. The bacterium was grown in batch culture at

different pH values for 14 days. Values between pH 6.5 and 7.1 are

the means (�SD) of three replicates. Values below and above this pH

range are single data.



Substrate utilization duringchemolithoautotrophic growth

The aim of this study was to examine the substrate utili-

zation of S. gotlandica GD1 in dependence of pH. The

chosen pHs values were pHs 7.1 (present pH in Baltic Sea

redox zones), and pHs 6.6 (the critical point, at which an

influence of pH on maximal cell numbers was visible).

Maximal cell numbers, and substrate utilization showed

no significant differences between the pHs 7.1 and 6.6 val-

ues (ANOVA, P > 0.05) (Fig. 3). Thus, the results of pHs

7.1 and 6.6 were summarized together. In all trials within

these 2 pHs values, a cell abundance of 1.4 9 107

� 2.7 9 106 cells mL�1 was reached in 9 days (Fig. 3).

However, at a pHs directly below 6.6 cell numbers were

sharply reduced (see Figs. 2, 3), for example at pHs 6.55

only a maximal cell number of 3.8 9 106 � 4.1

9 105 cells mL�1 at day 9 was achieved (Fig. 3C).

At pH 7.1 and 6.6 S. gotlandica GD1T completely con-

sumed the 1000 lmol L�1 thiosulfate, metabolizing most

of it to sulfate within the 9 days (1322.9 � 201

lmol L�1). Nitrate was only partially consumed and at

the end of the experiment still 236 � 129 lmol L�1 of

nitrate could be measured. In contrast, at pHs 6.55 the

bacteria used 742.5 � 391.8 lmol L�1 of nitrate and

824.3 � 144.0 lmol L�1 of thiosulfate and produced

903.8 � 373.0 lmol L�1 sulfate.

According to Figure 3 the exponential growth occurred

between days 3 and 9. During the late exponential phase

(day 5–9) differences in the substrate concentrations were

the most significant, thus reducing methodological biases

(which are larger when only small concentration changes

occur). Hence, in the late exponential phase S. gotlandica

GD1T used 68.1 � 12.3 fmol nitrate cell�1 and 43.7 �5.9 fmol thiosulfate cell�1 and produced 77.9 � 17.9

fmol sulfate cell�1 at pHs 7.1 and 6.6. The cellular growth

rate was at both pHs values around 0.4 � 0.01 h�1. In con-

trast, at pHs 6.5, the growth rate was 0.3 � 0.02 h�1, but

with strongly enhanced substrate turnover. In fact, the cells

used 414 � 151 fmol nitrate cell�1, 247 � 209 fmol thio-

sulfate cell�1, and produced 944 � 563 fmol sulfate cell�1

than cultures maintained at a higher pH.

The negative controls for all chemical analysis remained

constant during the incubation time and were always in

the same range as the standards samples of 0 lmol thio-

sulfate, nitrate or sulfate, respectively, used for calibration.

Hence, there was no evidence that other chemical com-

pounds influenced the measurements. The discrepancy

between thiosulfate utilization and sulfate production at

6.55 was probably caused by methodical errors. The mea-

surements of nitrate and thiosulfate at the beginning and

after 24 h confirmed the added concentrations of

1 mmol L�1 for each of these substrates. The pH could

be kept relatively constant with a decrease of pHe of

0.092 � 0.046 units.

Discussion

The primary aim of this study was to examine the

response of the e-proteobacterium S. gotlandica GD1T

toward changes in DIC and pH. This could be achieved

(A)

(B)

(C)

Figure 3. Anaerobic chemoautotrophic growth of Sulfurimonas

gotlandica GD1T in a batch culture at pH 7.1 (A), pH 6.6 (B), and pH

6.55 (C). Cell abundance and nitrate (electron acceptor), thiosulfate

(electron donor), and sulfate (formed by thiosulfate oxidation)

concentrations were quantified daily. (A–C) are the means of three,

two, and two replicates, respectively. Error bars are standard

deviations.



by assessing the impact of changes in DIC concentration

and pH on growth and maximal cell numbers of S. got-

landica GD1T in batch culture growth experiments.

Hydrogen sulfide is the major substrate in anoxic

waters and S. gotlandica GD1T seems to be primarily

responsible for hydrogen sulfide oxidation in the Baltic

Sea (Grote et al. 2012). However, at the oxic–anoxicinterface and the upper sulfidic zone thiosulfate concen-

trations are in a similar range as hydrogen sulfide concen-

trations (Bruckner et al. 2013) and thiosulfate serves as

an alternative substrate for S. gotlandica GD1T. We used

thiosulfate as electron donor in the experiments as

substrate concentrations can be much better controlled

compared to hydrogen sulfide. Although thiosulfate

seemed to be entirely consumed at the end of the experi-

ment (see Fig. 3), earlier experiments with this strain did

not produce higher cell numbers with higher thiosulfate

concentrations (Bruckner et al. 2013; Labrenz et al. 2013).

Therefore, other potentially limiting factors, related to cell

concentration, have to be considered, such as the accu-

mulation of inhibitory metabolic products.


The growth-stimulating effects of increasing DIC concen-

trations for phytoplankton are well documented (e.g.,

Iglesias-Rodriguez et al. 2008) whereas only few studies

were performed with chemolithoautotrophic bacteria.

According to the current DIC concentrations of about

2 mmol L�1 and 3.5 mmol L�1 (Frey et al. 1991; Beldow-

ski et al. 2010) in the redox zones of the Baltic and the

Black Sea, our results show that these DIC concentrations

are well within the range supporting maximal growth of

S. gotlandica GD1T and related epsilonproteobacteria. The

results suggest that a further increase in DIC concentra-

tion in the redox zones should have no additional direct

effect on these bacteria.

A higher DIC concentration in the ocean causes a shift

in the DIC speciation toward carbon dioxide, resulting in

a decrease of pH, that is at pH 7.1 90% of the DIC speci-

ation is hydrogen carbonate while at pH 6.3 the balance

shifts to 50% hydrogen carbonate and 50% carbon diox-

ide (Deffeyes 1965). However, this shift in speciation

should not have an influence on growth of S. gotlandica

GD1T as a DIC concentration of 800 lmol L�1 was

already sufficient to promote maximal cell numbers

(Fig. 1). Comparable saturation curves for increasing DIC

concentrations had been determined for other bacterial

and phytoplankton species. Clark and Flynn (2000)

described the relationship between the carbon-specific

growth and the DIC concentration of several marine phy-

toplanktons. Most of these species reached a saturation

between 500 and 1000 lmol L�1 DIC. Furthermore,

Dobrinski et al. (2005) showed that for the chemolitho-

autotrophic c-proteobacterium Thiomicrospira crunogena,

isolated from a hydrothermal vent, the half-saturation

DIC concentration was 220 lmol L�1 and saturation was

reached at 1000 lmol L�1 DIC, which is in the same

range as the values determined for S. gotlandica GD1T. In

contrast, Scott and Cavanaugh (2007) showed that the

chemoautotrophic Solemya velum symbionts reach satura-

tion at a CO2 concentration of 100 lmol L�1. However,

this saturation is also well within the range of the CO2

concentration in the environment, where S. velum was

collected. In addition, they could prove that these symbi-

onts rely on CO2 and not on bicarbonate.

Genomic data indicate that S. gotlandica GD1T is capa-

ble of using both CO2 and bicarbonate by converting

intracellular bicarbonate to CO2 with the carbonic anhy-

drase (Grote et al. 2012). In addition, Dobrinski et al.

(2005) could prove that the chemolithoautotrophic c-pro-teobacterium Thiomicrospira crunogena has the ability to

use both external CO2 and bicarbonate. Therefore, it

seems probable that S. gotlandica GD1T has also the abil-

ity to use both external CO2 and bicarbonate as inorganic

carbon source.


The pH range at which S. gotlandica GD1T grew well (pH

6.6–8.0) was relatively narrow compared to that of other

chemolithoautotrophic proteobacteria. For example several

c-proteobacterial Thiomicrospira species from hydrother-

mal vents grew at a wide pH range of 5.3–8.5 or 4.0–7.5(Brinkhoff et al. 1999). Also for other chemoautrophic

e-proteobacteria a relatively wide tolerable pH range was

found, for example for Sulfurimonas paralvinellae and

Sulfurimonas autotrophica, the pH range was 5.4–8.6(optimum 6.1) and 5.0–9.0 (optimum 6.5), respectively

(Inagaki et al. 2003; Takai et al. 2006). Sulfurimonas deni-

trificans, the closest cultivated relative of S. gotlandica

GD1T (Grote et al. 2012), has a pH optimum of 7.0 (Tim-

mer-ten Hoor 1975). Most of these investigated bacteria

with a more extended pH range compared to S. gotlandica

GD1 exist in habitats such as hydrothermal vents, where

pH changes are frequent and rapid. Thus, the extended pH

range suggests that it is an adaptation to the extremely var-

iable conditions, whereas S. gotlandica str. GD1 was iso-

lated from a relatively stable habitat. Scott and Cavanaugh

(2007) confirm this conclusion with their studies about

chemoautotrophic c-proteobacteria, living as endos-

ymbionts in sulfidic/oxic interfaces. These Solemya velum

symbionts have also a relatively narrow range of pH opti-

mum (between pH 7.4 and 8.5), showing the same sharp

decline in growth directly below and above these levels.



The substrate utilization did not differ significantly

between pHs 7.1 and 6.6. However, at a pHs below 6.6 the

situation changed drastically and growth of S. gotlandica

GD1 was obviously impaired, and consumption of nitrate

and thiosulfate was strongly reduced. Thus, the important

functional role of S. gotlandica GD1T in the redoxcline

nitrogen and sulfur cycles would probably be impacted.

While intracellular pH was not measured in this study,

it is supposed that the intracellular pH varies by around

0.1 units per unit change in the external pH (Hackstadt

1983). According to Booth (1985) changes in pH outside

the pH optimum leads to an inhibition of both enzyme

activity and cell growth (Booth 1985). However, thus far

the exact mechanism of intracellular pH regulation is not

completely understood. Earlier studies have shown that

the regulation is energy dependent and requires a high

respiratory rate (Booth 1985). Hence, the high substrate

utilization per bacterium at pHs 6.55 could be at least

partially explained by regulation of the intracellular pH

outside the optimum pH.

As substrate utilization in the batch culture experiments

was significantly higher than under environmental condi-

tions, the results rather reflect the carrying capacity of

S. gotlandica GD1T at the given conditions. Future studies

should aim to more accurately simulate in situ conditions

(e.g., with chemostat cultures). Due to global warming and

higher CO2 concentrations in the atmosphere an increase

in the DIC concentration and a decrease of pH by about

0.3 units in the ocean, known as ocean acidification, is pre-

dicted (Houghton et al. 2001). Our results suggest that a

direct impact of acidification on S. gotlandica GD1T and

related organisms should not be very strong, as the opti-

mum pH range for this model organism is still within the

range of the predicted changes in pH. On the other hand,

indirect effects on S. gotlandica GD1T might be more

important than direct ones. For example there is evidence

that nitrification, the process which delivers nitrate for

denitrifying bacteria, is negatively influenced by a decrease

in pH and an increase in pCO2 (Huesemann et al. 2002;

Denecke and Liebig 2003; Hutchins et al. 2009).

Acknowledgments

We are thankful for a grant from the German Federal

Ministry of Education and Research (BMBF), joint

research project BIOACID (project number: 03F0608F),

subproject 1.1.1. We also would like to thank Bernd

Schneider for his help and advice on the experimental

design.

Conflict of Interest

None declared.

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epsilonproteobacterium Sulfurimonas gotlandica GD1Toceanrep.geomar.de/26985/1/mbo3153.pdf · doi:10.1002/mbo3.153 Abstract Epsilonproteobacteria have been found globally distributed